Specific targeting antibacterial peptide aiming at clostridium perfringens as well as preparation method and application of specific targeting antibacterial peptide

Abstract

The invention provides a specific targeting antibacterial peptide aiming at clostridium perfringens as well as a preparation method and application thereof, and belongs to the technical field of bioengineering, and the sequence of the specific targeting antibacterial peptide aiming at clostridium perfringens is as shown in SEQ ID No.1. A peptide fragment WYSPQWWNHHRH with clostridium perfringens affinity obtained through screening of a phage display peptide library is used as a targeting peptide fragment, a broad-spectrum antibacterial peptide GKEFKRIVKWPWWPWRR is used as a functional region, the peptide fragment WYSPQWWNHHRH and the broad-spectrum antibacterial peptide GKEFKRIVKWPWWRR are connected through a flexible structure GGG to construct the antibacterial peptide with a clostridium perfringens targeting effect, the antibacterial peptide shows an excellent targeting effect, and under the condition that the influence on other bacteria is small, the antibacterial peptide with the clostridium perfringens targeting effect is achieved. And the compound has efficient antibacterial activity on clostridium perfringens. In addition, the specific targeting antibacterial peptide W1LI aiming at the clostridium perfringens has relatively low hemolytic activity and cytotoxicity.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a specific targeted antimicrobial peptide W1LI against Clostridium perfringens, its preparation method, and its application. Background Technology

[0002] Clostridium perfringens poses a particularly significant threat to livestock farming. For example, in broilers, the high detection rate of this bacterium in the intestines often leads to necrotic enteritis. Infected piglets are prone to hemorrhagic necrotic enteritis, with persistently high morbidity and mortality rates, severely impacting the pig industry. Currently, Clostridium perfringens exhibits widespread resistance to many commonly used antibiotics, including tetracyclines, lincomycin, macrolides, quinolones, aminoglycosides, and bacteriocins, rendering these drugs virtually ineffective. Although penicillin is considered the first-line treatment for anaerobic bacterial infections, its long-term use has led to widespread resistance, and its therapeutic efficacy against this bacterium remains uncertain. Therefore, developing novel antibiotic alternatives that can effectively inhibit Clostridium perfringens is urgently needed.

[0003] However, antimicrobial peptides are often similar to traditional antibiotics, being broad-spectrum antibacterial drugs. Using antimicrobial peptides as a treatment for intestinal infections faces significant challenges: broad-spectrum antimicrobial peptides may simultaneously eliminate both pathogens and host symbiotic bacteria, leading to microbiome disruption and adverse reactions. Therefore, developing drugs that can specifically identify and kill *Clostridium perfringens* for precise treatment of *Clostridium perfringens* infections and reduce disruption to the intestinal microecological environment is of great importance. Summary of the Invention

[0004] In view of the above shortcomings, the purpose of this invention is to provide a targeted antimicrobial peptide W1LI against Clostridium perfringens, which has the ability to selectively kill Clostridium perfringens.

[0005] The object of the present invention is achieved as follows: a targeted antimicrobial peptide W1LI against Clostridium perfringens, the sequence of which is shown in SEQ ID No.1.

[0006] Another objective of this invention is to provide a method for preparing the Clostridium perfringens-targeting antimicrobial peptide W1LI, as follows: A broad-spectrum antimicrobial peptide LI is used as the parent peptide, with the sequence GKEFKRIVKWPWWPWRR; Clostridium perfringens protein is extracted, and then Clostridium perfringens is co-incubated with a phage-displayed peptide library to screen for the targeting peptide sequence W1, which has affinity for Clostridium perfringens. The sequence WYSPQWWNHHRH is used as the target peptide sequence. Finally, a flexible structure GGG is used to connect these two sequences, resulting in the design of an antimicrobial peptide W1LI with selective inhibitory activity against the target bacterium, as shown in SEQ ID No. 1. The peptide is prepared using a solid-phase chemical synthesis method, and after purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, its antibacterial activity, hemolytic activity, and eukaryotic cytotoxicity are determined, ultimately naming it the antimicrobial peptide W1LI.

[0007] Another object of the present invention is to provide the use of the specific targeting antimicrobial peptide W1LI as described above in the preparation of a medicament for treating diseases caused by Clostridium perfringens.

[0008] Furthermore, the application of the drug is as a medicated feed additive.

[0009] Another object of the present invention is to provide a medicament suitable for treating Clostridium perfringens infection, said medicament containing the antimicrobial peptide W1LI as described above.

[0010] The beneficial effects and advantages of this invention are as follows: The specific targeted antimicrobial peptide W1LI of this invention exhibits extremely strong selective antimicrobial activity against *Clostridium perfringens* (MIC = 4 μM); it did not show significant antimicrobial activity against a series of Gram-positive and Gram-negative bacteria, including *Staphylococcus aureus*, *Lactobacillus rhamnosus*, and *Lactococcus lactis*. Notably, the hemolysis rate of erythrocytes induced by the specific targeted antimicrobial peptide W1LI at a concentration of 64 μM did not reach the 10% hemolysis threshold. At the same concentration, the survival rate of porcine small intestinal epithelial cells IPEC-J2 reached 98.6%. In summary, the specific targeted antimicrobial peptide W1LI is a highly efficient targeted antimicrobial peptide with strong targeting against *Clostridium perfringens* and high application value, showing potential as a substitute for feed antibiotics. Attached Figure Description

[0011] Figure 1 This is a high-performance liquid chromatogram of W1LI, a specific targeting antimicrobial peptide against Clostridium perfringens according to the present invention.

[0012] Figure 2 This is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MAMS) image of W1LI, a specific targeting antimicrobial peptide against Clostridium perfringens according to the present invention.

[0013] Figure 3 The diagram shows the hemolytic activity of the parent peptide LI, the specific targeting antimicrobial peptide W1LI against Clostridium perfringens, and the bee venom Melittin of the present invention.

[0014] Figure 4 The diagram shows the cytotoxicity of the parent peptide LI, the specific targeting antimicrobial peptide W1LI against Clostridium perfringens, and the bee venom Melittin in the examples. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0016] Example 1

[0017] Design of W1LI, a specific targeting antimicrobial peptide against Clostridium perfringens

[0018] In this embodiment, bacterial protein extraction kits were used to extract proteins from *Escherichia coli* K88, *Staphylococcus aureus* ATCC29213, *Salmonella paratyphimurium* CICC 21493, *Lactobacillus rhamnosus*, and *Clostridium perfringens*. First, the extracted proteins from *E. coli* K88, *Staphylococcus aureus* ATCC 29213, *Salmonella paratyphimurium* CICC 21493, and *Lactobacillus rhamnosus* were incubated with a phage-displayed peptide library for antigen shielding. Then, the *Clostridium perfringens* protein was incubated with the phage-displayed peptide library. After three rounds of screening, a targeting peptide W1 with affinity for *Clostridium perfringens* was selected, with the amino acid sequence: WYSPQWWNHHRH. The targeting peptide and the parent peptide LI were linked using a flexible structure GGG to design a novel peptide W1LI with strong targeted antibacterial activity against *Clostridium perfringens*. The above-mentioned antimicrobial peptide was synthesized using a peptide synthesizer via solid-phase synthesis. The amino acid sequence of the antimicrobial peptide W1LI is: WYSPQWWNHHRHGGGGKEFKRIVKWPWWPWRR. The sequence of the antimicrobial peptide is shown in Table 1.

[0019] Table 1. Amino acid sequence of W1LI, a specific targeting antimicrobial peptide against Clostridium perfringens.

[0020]

[0021] Example 2

[0022] Solid-phase chemical synthesis of W1LI, a specific targeted antimicrobial peptide against Clostridium perfringens.

[0023] 1. The preparation of antimicrobial peptide W1LI proceeds sequentially from the C-terminus to the N-terminus using a peptide synthesizer: First, Fmoc-X (where X is the first amino acid at the C-terminus of each antimicrobial peptide) is inscribed into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; next, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-trimethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide, and its side chain may be protected with groups such as Trt as needed) is mixed with coupling reagents and activators, and then reacts with the free amino groups on the resin to form peptide bonds. After the reaction is complete, the resin is thoroughly washed to remove excess reagents; this procedure is repeated sequentially from the C-terminus to the N-terminus until the synthesis is complete, yielding resin with the side chain protection removed by the Fmoc group;

[0024] 2. Add the cleavage reagent to the peptide resin obtained above, react at 20 ℃ in the dark for 2 h, and filter; wash the precipitate with TFA (trifluoroacetic acid), mix the washings with the above filtrate, concentrate by rotary evaporator, add about 10 times the volume of pre-cooled anhydrous diethyl ether, precipitate at -20 ℃ for 3 h, and a white powder will precipitate. Centrifuge at 2500 g for 10 min, collect the precipitate, wash the precipitate with anhydrous diethyl ether, and vacuum dry to obtain the peptide. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5.

[0025] 3. Equilibrate the column for 30 min using 0.2 M sodium sulfate (adjusted to pH 7.5 with phosphoric acid). Dissolve the peptide in 90% acetonitrile aqueous solution, filter, and elute using a C18 reversed-phase atmospheric pressure column with gradient elution (eluting agent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), at a flow rate of 1 mL / min and a detection wavelength of 220 nm. Collect the main peak and lyophilize it. Further purify using a reversed-phase C18 column with elution buffer A being 0.1% TFA / aqueous solution and elution buffer B being 0.1% TFA / acetonitrile solution, with an elution concentration of 25% B to 40% B, an elution time of 12 min, and a flow rate of 1 mL / min. Collect the main peak again and lyophilize it.

[0026] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weight (e.g., ...) shown in the mass spectrum is determined by the electrospray ionization mass spectrometry method. Figure 1 , 2 The molecular weight (as shown in Table 1) is basically consistent with the theoretical molecular weight, and the purity of the antimicrobial peptide is greater than 95%.

[0027] Example 3

[0028] 1. Determination of antibacterial activity: The antimicrobial peptide W1LI was prepared into a stock solution for use. The minimum inhibitory concentration (MIC) of several antimicrobial peptides was determined using the microbroth dilution method. A series of antimicrobial peptide solutions were prepared using a two-fold dilution method with 2 mg / ml BSA (containing 0.01% acetic acid) as the diluent. 100 μL of each solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial culture (~10 μL) was added to each well. 5 (Cells / mL) were added to each well. A positive control (containing bacterial culture but no antimicrobial peptides) and a negative control (containing neither bacterial culture nor antimicrobial peptides) were also included. After incubation at 37 ℃ for 14-18 h, the OD values ​​were... 492nm The minimum inhibitory concentration (MIC) is the antimicrobial peptide concentration that inhibits the growth of 99.9% of microorganisms when absorbance is measured. The test results are shown in Table 2.

[0029] Table 2. Minimum inhibitory activity of the specific targeted antimicrobial peptide W1LI against pathogenic microorganisms.

[0030]

[0031] As shown in Table 2, LI exhibits broad-spectrum antibacterial activity, demonstrating high antibacterial activity against most Gram-negative and Gram-positive bacteria. However, the specific antimicrobial peptide W1LI targeting *Clostridium perfringens* only shows antibacterial activity against *Clostridium perfringens*, exhibiting minimal antibacterial activity against other non-target strains, demonstrating precise targeting specificity.

[0032] 2. Determination of hemolytic activity: 1 mL of fresh human blood was collected, anticoagulated with heparin, and dissolved in 2 mL of PBS solution. The mixture was centrifuged at 3000 rpm for 10 min, and red blood cells were collected. The red blood cell suspension was washed three times with PBS solution and then resuspended in 10 mL of PBS solution. 50 µL of the red blood cell suspension was mixed thoroughly with 50 µL of antimicrobial peptide solutions of different concentrations and incubated at 37 ℃ for 1 h. After incubation, the mixture was removed and centrifuged at 4 ℃ at 3000 rpm for 10 min. The supernatant was then measured at 570 nm using a microplate reader. 50 µL of red blood cells with 50 µL of PBS solution served as a negative control, and 50 µL of red blood cells with 50 µL of 0.1% Tritonx-100 served as a positive control. The minimum hemolytic concentration was the concentration of the antimicrobial peptide that caused a 10% hemolysis rate. The test results are shown in […]. Figure 3 .pass Figure 3 It can be seen that W1LI did not exhibit hemolytic activity within the detection range, and failed to induce 10% hemolysis of erythrocytes at a concentration of 64 µM, showing a significant difference from the control group bee venom.

[0033] 3. Determination of eukaryotic cytotoxicity: The MTT assay was used to detect the cytotoxicity of IPEC-J2 porcine small intestinal epithelial cells. The specific steps are as follows:

[0034] (1) Preparation of culture medium and cell culture: After the pig small intestinal epithelial cells IPEC-J2 frozen in liquid nitrogen were thawed, they were inoculated into a culture medium containing 10% fetal bovine serum and 1% double antibiotics. They were passaged at 37℃ and 5% CO2 until the cells reached 80%-90% of the bottom of the bottle.

[0035] (2) Cell processing: The cells were washed and resuspended three times with sterile PBS, and then digested with 0.25% trypsin solution to detach them from the bottom of the flask. The cells were then washed with complete culture medium to obtain a single-cell suspension. At the same time, a final concentration of approximately 2 × 10⁻⁶ cells was filled into a 96-well plate. 4 50 μL of cell suspension.

[0036] (3) Antimicrobial peptide treatment: Add 10 μL of antimicrobial peptide to the first well of a 96-well plate and serially dilute it. Add 50 μL of diluted cell suspension to wells 1-10 of the 96-well plate. Add 50 μL of complete culture medium to well 11 and 100 μL of complete culture medium to well 12. Incubate at constant temperature for 4 h.

[0037] (4) Toxicity test: Add 50 μL of 5 mg / mL MTT solution to a 96-well plate, continue culturing for 3-4 hours, then add 150 μL of DMSO, and measure the OD value using a microplate reader. 570 The absorbance was measured at nm, and the results are shown in [reference needed]. Figure 4 .pass Figure 4 It can be seen that the antimicrobial peptide W1LI did not show toxicity to porcine small intestinal epithelial cells IPEC-J2 within the detection range, and showed a significant difference from the control group melittin bee venom.

Claims

1. A specific targeting antibacterial peptide W1LI against Clostridium perfringens, characterized in that, The amino acid sequence is shown as SEQ ID No.

1.

2. The preparation method of the specific targeting antibacterial peptide W1LI against Clostridium perfringens according to claim 1, characterized in that, The method is as follows: taking broad-spectrum antibacterial peptide LI as the parent peptide, the sequence of which is GKEFKRIVKWPWWPWRR; and through extraction of Clostridium perfringens protein, then incubating Clostridium perfringens with phage display peptide library, screening out the target peptide sequence W1 with Clostridium perfringens affinity, the sequence of which is WYSPQWWNHHRH, finally connecting the two sequences with flexible structure GGG, obtaining the antibacterial peptide W1LI with selective inhibitory effect on the target bacteria, the sequence of which is shown as SEQ ID No. 1; The polypeptide is prepared by solid-phase chemical synthesis, and after reversed-phase high performance liquid chromatography purification and mass spectrometry identification; and after determination of antibacterial activity, determination of hemolytic activity and determination of eukaryotic cell toxicity, the polypeptide is finally named as antibacterial peptide W1LI.

3. Application of the specific targeting antibacterial peptide W1LI against Clostridium perfringens in the preparation of a drug for treating Clostridium perfringens infectious diseases according to claim 1.

4. Use according to claim 3, characterized in that: The application in the drug is the application in a drug feed additive.

5. A medicament suitable for the treatment of Clostridium perfringens infection, characterized in that: The drug contains the specific targeting antibacterial peptide W1LI against Clostridium perfringens according to claim 1.

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